Ner

ve C

ells

– M

aste

rs o

f C

om

mu

nic

atio

n The human nerve system consists of approximately one hundred billion nerve cells – give ortake a few billions. And each one of these cells has the ability to make contact withthouands of neighboring cells. Tiny membrane bubbles, the so-called vesicles, set freespecial messengers that affect the behavior of adjacent cells. To study what happens duringthese processes in detail on the molecular level is one of the topics of research at theinstiute. After all, these processes are the foundation for the nerve cells’ ability to collect andprocess information – including such complex brain functions as learning and memory.

The widely ramified exten-sions of a nerve cell (blue)are scattered with contact

points with other nervecells (red).

departments and research groups 57

Dr. Ralf Schneggenburger

Ralf Schneggenburger studied biology at the Universities of Göttingen and

Tübingen, and conducted part of hisgraduate studies at the University of

Seville in Spain. In 1993, he completed his Ph.D. thesis at the Max Planck

Institute for Biophysical Chemistry. He then went as a postdoctoral fellow to the University of the Saarland, and to the

Ecole Normale Supérieure in Paris, France. In 1996, he joined the Department of

Membrane Biophysics at the Max PlanckInstitute for Biophysical Chemistry. In 2001, he received a Heisenberg

fellowship from the DeutscheForschungsgemeinschaft, and,

since then, has been leading the Synaptic Dynamics and

Modulation research group.

rschneg@gwdg.de

www.mpibpc.mpg.de/abteilungen/140/groups/sdm/

Synaptic Dynamics and Modulation

Nerve cells in our brains communicate at specialized con-

tact sites, the synapses. During the process of chemical

synaptic transmission, an electrical impulse leads to the

opening of Ca2+ channels in the nerve terminals of the presynaptic

(signaling) cell. The resulting Ca2+ influx causes the fusion of vesi-

cles with the plasma membrane, and the ensuing release of neuro-

transmitter from the presynaptic cell. The neurotransmitter in turn

activates receptor-ion channel complexes on the postsynaptic

(receiving) nerve cell. Interestingly, repetitive electrical activity in

the presynaptic nerve cell leads to plastic changes in synaptic

strength over short periods of time. The responses in the post-

synaptic nerve cell either increase or decrease over time. This

dynamic behavior of synapses, or short-term synaptic plasticity,

influences the way in which the information flow in neuronal net-

works is integrated over short time periods.

A deeper understanding of the mechanisms of short-term plasticity

requires studying the signaling steps in the presynaptic nerve ter-

minal. Such studies are, however, hampered by the small size of

most types of nerve terminals. We therefore study an unusually

large glutamatergic nerve terminal, the calyx of Held, at which

presynaptic whole-cell patch-clamp recordings can be obtained.

This allows us to stimulate the nerve terminal electrically in the

voltage-clamp mode or chemically by elevating the intracellular

Ca2+ concentration by the photolytic release of ›caged Ca2+‹, caused

by a short light flash. The resulting increase in Ca2+ concentration

is measured by imaging the fluorescence of a Ca2+-sensitive indica-

tor dye (see image). We expect the accessibility of the calyx of Held

nerve terminals to such manipulations to produce new insights

into the signaling mechanisms that underlie synaptic transmission

and its short-term plasticity.

A series of fluorescent images of a calyx of Held is used to measure the increase of intracellular Ca2+ concentration, produced by Ca2+ uncaging witha short light flash. The right panel also shows the patch pipette used to measure the electrical signals in the presynaptic nerve terminal.

Meyer, A. C., E. Neher and R. Schneggenburger:Estimation of quantal size and number of func-tional active zones at the calyx of Held synapseby nonstationary EPSC variance analysis. J.Neurosci. 21, 7889–7900 (2001).

Felmy, F., E. Neher and R. Schneggenburger:Probing the intracellular Calcium sensitivity oftransmitter release during synaptic facilitation.Neuron 37, 801–811 (2003).

Wölfel, M., and R. Schneggenburger: Presynap-tic capacitance measurements and Ca2+

uncaging reveal sub-millisecond exocytosiskinetics and characterize the Ca2+ sensitivity ofvesicle pool depletion at a fast CNS synapse. J.Neurosci. 23 (2003)

58 departments and research groups

Professor Reinhard Jahn

After studying biology and chemistry,Reinhard Jahn obtained his Ph.D. fromthe University of Göttingen in 1981.Subsequently, he conducted research atthe Rockefeller University in New York(1983-1986) and the Max Planck Institutefor Psychiatry (presently renamedNeurobiology) in Munich (1986-1991).After this, he was appointed Professor of Pharmacology and Cell Biology at Yale University, New Haven, with a jointappointment at the Howard HughesMedical Institute. Since 1997, he has been a director at the Max PlanckInstitute for Biophysical Chemistry andhead of the Department of Neurobiology.Reinhard Jahn is also Honorary Professorof the University of Göttingen. In 1990,he received the Max Planck ResearchPrize, and was awarded the GottfriedWilhelm Leibniz Prize in 2000.

Internal research groups:Dr. Dirk FasshauerDr. Ulrich KuhntDr. Thorsten Lang

rjahn@gwdg.de

Neurons communicate with other cells by releasing neuro-

transmitters stored in synaptic vesicles in nerve termi-

nals. Upon excitation, a few vesicles fuse with the plasma

membrane. Like several other groups in our institute, we are inter-

ested in the molecular basis of the underlying membrane fusion

event.

Membrane fusion is not limited to neurons. Every eukaryotic cell is

compartmentalized into membrane-bound organelles that are con-

tinuously reorganized in a dynamic manner. Membrane-enclosed

transport vesicles are generated from precursor membranes and are

then transported to their destination where they fuse with intra-

cellular target membranes.

Most intracellular membrane fusion events are mediated by evolu-

tionarily conserved proteins. Among these, the SNARE proteins are

the best candidates for catalyzing the fusion reaction. The first

clues to their function were obtained from the study of powerful

bacterial toxins, botulinum neurotoxins and tetanus toxin. Al-

though tetanus and botulism affect different neurons, the toxins

share a common mechanism of action. They cut SNARE proteins in

half, which results in an inhibition of vesicle fusion and thus of

neurotransmitter release.

SNAREs are abundant on intracellular membranes and readily form

stable complexes. Each fusion reaction requires the cooperation of

several SNARE proteins. Furthermore, the SNAREs mediating differ-

ent intracellular fusion reactions are not identical, although some

SNAREs participate in multiple fusion steps. According to our

working model, the SNAREs operate as »nanomachines«. When

membranes get close to each other, the SNAREs engage each other

and »zipper up« from their distal ends towards the membrane.

Since energy is released during this reaction, the membranes are

forced into approximation, thereby initiating membrane fusion.

After fusion, the SNAREs have released their energy and need to be

Neurobiology

www.mpibpc.mpg.de/abteilungen/190/

Vesicles (green), bound to a sheet of plasma membrane containing SNAREclusters (red) are caught in the act of exocytosis. The vesicles are filled with apeptide linked to a green fluorescent protein. Upon fusion, the peptide isreleased and disappears.

Sutton, B., D. Fasshauer, R. Jahn and A.T.Brünger: Crystal structure of a SNARE complexinvolved in synaptic exocytosis at 2.4 Å resolu-tion. Nature 395, 347–353 (1998).

Takamori, S., J.S. Rhee, C. Rosenmund and R.Jahn: Identification of a vesicular glutamatetransporter that defines a glutamatergic pheno-type in neurons. Nature 407, 189–194 (2000).

Fasshauer, D., W. Antonin, V. Subramaniamand R. Jahn: SNARE assembly and disassemblyexhibit a pronounced hysteresis (2002) NatureStruct. Biol. 9, 144–151 (2002).

Lang, T., M. Margittai, H. Hölzler and R. Jahn:SNAREs in native plasma membranes areactive and readily form core complexes withendogenous and exogenous SNAREs. J. CellBiol. 158, 751–760 (2002).

Jahn, R., T. Lang and T.C. Südhof: Membranefusion. Cell 112, 519–533 (2003).

departments and research groups 59

disentangled with the assistance of chaperone pro-

teins and energy input.

Little is known how SNARE proteins are regulated,

i.e., how they are activated or silenced. SNARE pro-

teins interact with a long and still growing list of other

proteins that regulate their conformation and control

their availability for fusion reaction, particularly with

regard to regulated exocytosis. Several of these pro-

teins are as essential for fusion as the SNAREs, but for

most of them it is not yet known how they function at

the molecular level.

We aim to find out how the SNAREs manage to fuse

membranes, as well as how they are regulated by

other proteins. To this goal, we investigate the 3D

structures and the conformational changes of

SNAREs. Furthermore, we fuse artificial and native

membrane vesicles in the test tube. We have also

established cell-free but semi-intact vesicle-plasma

membrane preparations that retain the capacity

for regulated exocytosis. With the help of these

approaches, it is possible to learn more about the

multiple factors involved in SNARE-mediated mem-

brane fusion. Moreover, we collaborate with the

groups of E. Neher, C. Rosenmund, and J. Klingauf to

elucidate the effects of molecular changes on exo-

cytosis in intact cells and neurons. Finally, we are also

interested in the mechanisms by which synaptic

vesicles sequester and store neurotransmitters.

Three-dimensionalstructure of the SNAREcomplex mediatingneuronal exocytosis.The cleavage sites ofthe different botulinum(BoNT) and tetanus(TeNT) neurotoxins areindicated by arrows.

Working model showing how SNAREs (red, green, blue) drive membrane fusion by operating as nanomachines thatpull membranes into close promixity

60 departments and research groups

Dr. Christian Rosenmund

Christian Rosenmund studied pharmacy at the University of Frankfurt/Main andreceived his Ph.D. in physiology from theVollum Institute in Portland, Oregon, in1993. After working at the Salk Institute in La Jolla, California, for two years, hereturned to Germany as a Helmholtz fellowin 1995. Since 1998, he has been head of the Molecular Mechanisms of SynapticTransmission research group in theDepartment of Membrane Biophysics at the Max Planck Institute for Biophysical Chemistry. In 1999, Christian Rosenmund obtained his ›Habilitation‹ in physiology and is a Heisenberg fellow since then.

crosenm@gwdg.de

The brain is clearly our most complex organ; hundreds of

billion neurons process information from the outer world

to control – along with what we have learned in the past –

our second-to-second actions. Neurons use specialized junctions,

the synapses, for cell-to cell communication. The synapse trans-

duces the electrical activity of the sending, presynaptic neuron via

the Ca2+-triggered release of neurotransmitter (NT) filled vesicles.

The postsynaptic, receiving site transduces the NT signal into an

electrical signal via activation of ion channels. Multiple events

must occur at the presynapse before NT can be released, because

synaptic vesicles need to be filled with NT, tether to specific

release sites and be primed to reach fusion competence.

We are interested to learn at which stage of release presynaptic

proteins act, and how they function. In recent years, we have

started to functionally characterize mice bearing deletions of

(knockouts), or carrying mutations within presynaptic proteins

(knockins). We use cultured neurons to characterize the conse-

quences of these mutations on synaptic transmission with standard

patch-clamp electrophysiology. We further study the function of

the protein by test-

ing the ›rescue‹

ability of mutated

protein versions

when they are over-

expressed in knock-

out mice.

Among the exam-

ined molecules, we

identified and char-

acterized a protein

family involved in

the vesicle priming

(munc13s), and rec-

ognized them as

key regulators of

synaptic short-term

plasticity. We also

studied mutants of synaptotagmin-1 that confirm its role as a puta-

tive Ca2+-sensor of release. Neurons lacking the protein family of

complexins show reduced NT release efficiency accompanied by

desynchronisation of release, indicating an important role of these

proteins in Ca2+-triggered release. Our long-term goal is to achieve

a molecular and functional model of the release process at the

central synapse.

Mechanisms of SynapticTransmission

www.mpibpc.mpg.de/abteilungen/140/groups/mmcsf/

Many specific proteinsare necessary to fill thevesicles and to let themcontact and fuse withthe cell membrane.

Rosenmund, C., A. Sigler, I.Augustin, K. Reim, N. Brose, andJ.S. Rhee: Differential control ofvesicle priming and short termplasticity by Munc13 isoforms.Neuron 33, 411–424 (2002).Rhee, J.S., A. Betz, S. Pyott, K.Reim, F. Varoqueaux, I. Augustin,D. Hesse, T.C. Südhof, M.Takahashi, C. Rosenmund, andN. Brose: β-phorbol ester- anddiacylglycerol-induced augmen-tation of neurotransmitterrelease from hippocampal neu-rons is mediated by Munc13sand not by PKCs. Cell 108,121–133 (2002).Fernandez-Chacon, R., A.Konigstorfer, S.H. Gerber, J.Garcia, M. F. Matos, C.F. Stevens,N. Brose, J. Rizo, C. Rosenmund,and T.C. Südhof: SynaptotagminI functions as a calcium regulatorof release probability. Nature410, 41–49 (2001). Reim, K., M. Mansour, F.Varoqueaux, H.T. McMahon, T.C.Südhof, N. Brose, and C. Rosen-mund: Complexins regulate alate step in Ca2+-dependent neu-rotransmitter release. Cell 104,71–81 (2001).

departments and research groups 61www.mpibpc.mpg.de/abteilungen/140/groups/most/

Dr. Jürgen Klingauf

Jürgen Klingauf studied biology andphysics in Hamburg and Bonn. Hesubsequently spent three years at

Stanford University, California, as aBoehringer Ingelheim Fonds fellow,

and received his Ph.D. in Physics from the University of Göttingen in 1999.

Since 2000, he has been head of theMicroscopy of Synaptic Transmission

group in the Department of Membrane Biophysics at the

Max Planck Institute for Biophysical Chemistry.

jklinga@gwdg.de

Pyramidal nerve cell from rat brain in cell culture. On the right, the smallsynaptic boutons (∼∼ 1/1000 of a millimeter in diameter) are specifically markedwith a fluorescent dye.

ing the entire process, from vesicle transport

through fusion with the membrane to vesicle

recycling. Although single vesicles are far too

small to be visualized by standard light

microscopy, their fluorescence signals reveal a

lot of vital information.

When nerve cells transmit a signal, this happens in

specialized contact zones, the so-called synapses.

There, small membrane bubbles, the synaptic vesi-

cles, fuse with the cell membrane and release a transmitter sub-

stance. If a nerve cell ›has something to say‹, a few dozen of those

vesicles will be fused within a few seconds. The membrane needs

to be recycled swiftly – otherwise the synapse would swell up and

the supply of release-ready vesicles would disappear. Thus, the

membrane of fused vesicles is invaginated, pinched off, and

rearranged in new vesicles that are eventually refilled with trans-

mitter substance.

How this recycling process works, however, is not yet understood

in molecular detail. Based on electron microscopic investigations,

several different mechanisms are being discussed. Such images,

however, provide only snapshots of the fast events occurring in

living cells. Variants of fluorescence microscopy techniques devel-

oped at this institute promise to open up new avenues for viewing

these processes in real time. With these tools, we want to ›spy out‹

some of their dynamic properties.

Our objects of study are nerve cells of rat and mouse brain, which

we can keep in cell culture for a number of weeks. When placed in

culture dishes, freshly isolated nerve cells re-grow and even form

new synaptic connections with each other. These we observe dur-

Electron microscopic picture of a synapse packed withnumerous synaptic vesicles.

Microscopy of SynapticTransmission